Essentialsin "

_$06S$

Poster Presentation

FASEB/ASBMB Symposium - April, 1991Poster # 2947

The Rapid Isolation of Highly PurifiedCadmium Metallothioneins Using HPLC

G. Vella*, J. Bylandert, D. Hazen-Martint, K. O'Connor*,J. Krol* & C. Phoebe, Jr*.

*Millipore Corporation, Waters Chromatography Division34 Maple Street, Milford, MA. 01757 U.S.A.

tDepartment of Pathology, Medical University ofSouth Carolina, Charleston, SC. 29425 U.S.A.

Waters. The absolute essential WatersChromotographyDivisionMillipore Corporation Woters '}for bioresearch. 34 Maple St. Divisionof MIU.IPORE

Milford, MA O1757(508) 478-2000

41__ 81

INTRODUCTION:

The central role of the proximal tubule in the renal toxicity due to cadmium (Cd)poisoning has been well documented. However, the mechanisms leading to theonset of this nephrotoxic effect are presently not well understood. The most likelymechanismfor the initiation of toxicity involves the accumulation of Cd in the liverwhere it induces the formation of, and is complexed with the metallothioneins (MT-Iand MT-III low molecular weiaht bindina proteins, followed by release from the liver

" " ¥ " - " " rand subsequentabsorption by the proximal tubule. Methods for obtaining MTs fospecific RIA and immunohistochemistry have been hampered by difficulties inremoving impurities, particu!arly, from tl_e.more immunogenic of the two Cd-bindingproteins, Cd-MT-I. To obtain highly purified Cd-MTs for use in RIA as well as toassay the relative affects of exposure to Cd-MT and ionic Cd in vitro on humanproximal tubule cells, Cd-MT-Iand Cd-MT-IIwere preaared from rat liver for use inihese.studiesusing a two step chromatoqraphic procedure employing traditional _elfiltrahon followec[ by a rapid h_gh performance anion exchange chromatographicstep. The two main fractions collected were assessedfor homogeneity,by analyticalanion exchange, gel filtration (GFC) and reverse phase htgh performancechromatography. The fractions were also assayed for cadmium by atomicabsorbance spectroscopyand by high performance liquid chromatography.

SAMPLEPREPARATION.

Liver homogenatescontaining Cd-MT-Iand Cd-MT-IIwere prepared as described byWinge et al (1). Essentially, rats injected subcutaneously with cadmium chloridewere sacrificed, the livers excised and pooled and homogenized with 0.001 Mpotassium phosphate buffer, pH 7.8. The homogenates were centrifuged to yieldparticle-free supernatant fluids which were heate_ at 60 ° C for 10 minutes. Ther'esultingcoagulated material was removed by centrifugation. The supernatant fluidswere concentrated by lyophilization and chromatographed on a Sephadex G-75column eluted with 0.01 M potassium phosphate BufFer, pH 7.8. The cadmiumcontaining fractions were pooled and further purified by high performance anionexchange chromatography.

CHROMATOGRAPHIC RESULTS:

Figure 1 showsa high,performance anion exchange chromatogram in which the real-hmeabsorbance ratio s (250nm/280nm) of 24-28 (higher than previously reported)were used to differentiate Cd-MT-Iand Cd-MT-IIfrom other proteins present in thesample. Repeated anion exchange separations revealed a time dependentconversion of a non-metallothionein protein component to form which coeluted withCd-MT-1 (Figure 2). The formation of the interfering protein was eliminated bykeeping the sample at refrigerated temperatures and utilizing a fast highperformance anion exchange p.uri!ication step. Figure 3 illustrates the preparativepurification of the crude mater_al _nwhich two fractions corresponding to Cd-MT-I

":ql

and Cd-MT-II were collected. The overall time for this purification step was 30minutes. Figures4-6 show the chromatoqraphic evaluation of the collected fractionsby anion exchange, gel filtration and" reverse phase high performance liquidchromatography. In order to determine the concentration of cadmium present in thecollected fractions, samples of each were submitted for analysis by atomicabsorption spectroscopy as well as HPLCanalysis for transition metals. Figure 7shows the separation oF nine standard transition metals by HPLC. The fractions aswell as the crude GFC material were first diluted with 50 mM nitric acid followed

by passage through a C18 Sep-Pak® Plus cartridge. Analysis of the fractionsdemonstrated the presence of cadmium as well as zinc (Figure 8). These results(Figure 8).correlate well with those observed with AA and show a constantcadrnlum/zlnc ratio with the anion exchange purification step.

CONCLUSIONS:

The anion exchange purification methodology has demonstrated the importance ofspeed which has p.ermi!tedthe isolation of highly purified Cd-MT's in high yields.Due to the sensitivity, th_smethod may also be amenable as an alternative for a non-

radioactive assay of Cd-Mt formation and. breakdown, in toxicolo,gical, studies. It istalso demonstrated that the HPLCanalys_sof transition metals _sconsistent with thaof AA and additional information can be obtained in a single run.

REFERENCES:1. Winge, D.R., Premakumar,R., and Rajagopalan, V.K. (1975) Arch. Biochem. Biophys. 170, 242-252.

CHROMATOGRAPHIC CONDITIONS

ANALYTICAL ANION EXCHANGE CHROMATOGRAPHY

Column: Protein-PakTM DEAE8HR in a glass AP-1 column (10 x 100 mm)Method: AutooBlendTM TrisInitialConditions: 20 mM Tris, pH 8.5FinalConditions: 20 mM Tris, pH 8.5 plus 50 mM Sodium ChlorideGradientDuration: 20 minutes,linearFlow Rate: 1.5 ml/minDetection: 250 nm, 280 nm and Ratio 250/280Instrumentation: 650 Advanced Protein Purification System and 490 Programmable Multi-

Wavelength UV/Visible Detector

PREPARATIVEANION EXCHANGE CHROMATOGRAPHY

Column: Protein-PakDEAE8 HR in a glass AP-5 column (50 x 100 mm)Method: AutooBlend Tris (as above)Flow Rate: 37.5 ml/min.Detection: 250 nmInjection Vol.: 50 mlInstrumentation" 650 Advanced Protein PurificationSystem and 484 Tunable UV/Visible

Detector with a non-metallic flow cell

REVERSEPHASE CHROMATOGRAPHY

Column: Delta-PakTM HPI (3.9 x 150 ram) C18, 5 l_m, 300,_,EluentA: Water with 0.1% TFAEluentB: Acetonitrile with 0.1% TFAGradient: 0% B to 40% B in 30 minutes (linear) hold for 5 minutesFlow Rate: 0.5 ml/min.ColumnTemp.: 30° CDetection: 214 nmSystem: 625 Non-Metallic HPLCSystem with 994 Photodiode Array Detector

GEL FILTRATIONCHROMATOGRAPHY

Columns: Protein KW-802.5, 803, and 804Eluent: 50 mM Sodium Phosphate,pH 7.0 with 300 mM NaCIFlow Rate: 0.5 ml/minDetection: 250 nmSystem: 650 Advanced Protein Purification System and 490 Programmable Multi-

Wavelength UV/Visible Detector

TRANSITION METAL ANALYSIS CHROMATOGRAPHY

Column" Delta-Pak C18, 5 llm, 100,_, (3.9 x 150 mm)Eluent: 2 mM Sodium Octane Sulfonate and 35 mM Tartrate, pH 3.65 with 5%

AcetonitrileFlow Rate: 0.8 ml/minDetection: Post-columnPARat 0.4 ml/min, monitored at 500 nmSystem" ActiON Analyzer Non-Metallic System, Reagent Delivery Module and 490

Programmable Multi-Wavelength UV/Visible Detector

Methods Developmen -Anion ExchangeChromatography ol Crude Material

0.6

v _ Cd-MT-I MT-IIC:: EO c-

_o0_")

._Q C_I

< j0 I 1 i I I i I

0.020

11)UmE13 (:_o

..Q C',I -- _

0 i i f i l I w

M

o 30 1 q

cO_N _ Column" Protein-PokTM DEAE 8HR_ 2O0It)e,l

o 10

--T 1" ) ! w w 1-r ! I l I w i i T-I I I [ W _ I l I I I I I [ "l I 1 I _' I _ I I | 1 1 I 1 1 T I 1 I I I m W [ _ W ! I ; I W 1 T I I 1 I 1 I [ I W !

0 5 10 ].5 20 25 30 35

,",_in,.,tes Figure 1

Analysis of Sample Degradation byAnion Exchange Chromatography

0.6

E Cd-MT-IIC

O

c_ Old Samplegc-O

Cd-MT-Io

0 I I I I-- I I I .....

Column: Protein-PakTM DEAE 8HR

0.15

E

O

_ FreshSampleU

o

0 5 10 15 20 25 30 35

_in0t,_s Figure 2i-

Preparative Anion Exchange Chromatography

_..8 - Column" Protein-PakTM DEAE 8HR

Cd-M1"-11Fraction

EIo '1.2

¢_ Cd-MT-I FractionUE

O

.<

0.6

o.oJ TMI I I I I I I | I I I w I I I I I I I w I I w I w I i I w w I I w i I w I I ; I I I I I w I I I I I ! I w I I i _ I I I I a I I I I I i I I w

0 5 :l.O _.5 20 25 30 35

Minutes Figure 3

Analysis of Fractions byAnion ExchangeChromatography

0.6

E Column: Protein-PakTM DEAE 8HRc

O

¢Xl

Cd-MT-I Fractionuc-O

..QI,,,.

O "-

0 i I I I I I i

_..0

E¢,.

o - Cd-MT-II Fraction¢Xl

u¢-O

..QO

_Q.<

0 5 10 15 20 25 30 35

Minutes Figure 4

Analysis of FractionsbyGel FiltrationChromatography

0.012 _

u_ - d-MT-I Fractionc E Columns"Protein KW-802.5, 803O c"

o _ and 804OI.O

__c',l.< . /

0 -- --

I I I I

0.008tl)u - H-MT-IIFraction,'-ED r-

_0_e,l -

/0 1 I 1 I

Analysis of Fractions byReversePhase Chromatography

0.4 --

¢)U,-Eu _ Cd-MT-I Fraction

_1_ ._. -O'--

0 '_ I "_"1 1 1 I 1 l

0.25 -

u¢_ Column: Delta-Pak HPI C18,C::: I:::: _a ,-- Cd-MT-II Fraction 5 gm, 300/k

..Q -

0 _ v --1 i I i f I

Transition Metal Analysis byReverse Phase Chromatography

0.3 - 2 Peak ID's: Conc. (ppm) Column" Delta-Pak C18,1 Fe+3 2.0 5 l_m, IOOA- 2 Cu 0.53 Pb 1.0

4 4 Zn 0.55 Ni 1.0 9

- 6 Co 0.57 Cd 1.0

- 8 Fe+2 2.0E 9 Mn 1.0r-- __ 5

Oo 6

u(...O

._O

o 8<(

3

L /1

--- i , - , , ,---r--r---r--'r--l_l , , Fq---rr--'r---v 1 r- , , , , , , , , I ' ' ' ' ' ' ' ' ' [- ' ' ' ' ' ' ' " ' '

0 5 _.0 :1.5 20

Minotes Figure 7

L.

Transition Metal Analysisof Fractionsand Crude

0.1 - Zinc

- Column" Delta-Pak C18,Cd-MT-I Fraction 5 gm, 100A_ Cadmium

/LI 1 I I

E 0.1-t-

OOLn Cd-MT-II Fraction

U¢-

O - _ _..Q._ I I I 1

Sample Zinc* Cadmium* Cd/Zn Cd by AA*Crude 14.91 44.23 2.97 46.0

0.1 _ Cd-MT-I 1.72 5.38 3.13 6._1Cd-MT-II 3.26 9.23 2.83 10.8

Crude * Results as gg/ml (ppm)

-__2_, J,_ ]_,,

0 5 I0 15 20

Minutes Figure 8

ANALYSIS OF TRANSITION METALSUSING DYNAMICALLY COATED

REVERSE PHASE COLUMNS ANDPOST COLUMN DERIVITAZATION

by Jim Kroi

Ion Analysis GroupMilford, Mass 01757508/478-2000 x3043

ANALYSIS OF TRANSITION METALS USING DYNAMICALLY COATEDREVERSE PHASE COLUMNS AND POST COLUMN DERIVATIZATION

Work by Cassidy in the late 1970°sshowed that HPLC reverse phasecolumns could be modified with alkyl sulfonate salts to yield columnswith cation exchange character. Reverse phase columns generallyexhibit higher column efficiencies than cation exchange resins. Thisincreased efficiency gives sharper, narrower peaks and hence betterdetection limits. The dynamically coated concept is used to separatedivalent transition metals such as Mn, Fe, Co, Ni, Cu, Zn, Cd, and Pb.

Octane sulfonate (OS) present in the eluent will saturate into the columnafter a short equilibration time. Since it is not permenantly bonded to thecolumn, any loss of OS with time and due to the sample matrix will bereplenished by the eluent. The concentration of the OS can be variedfrom 1 to 10mM to meet the cation capacity needs of the sample matrix.Data shows that retention of transition metals increase with increasingOS concentrations.

Normally cations are eluted from cation exchange columns using H+ orother cations competing for the site. This works well for alkali andalkaline earth cations but is not effective for transition metals. However,transition metals will chelate with various organic acids making themanionic or neutral metal complexes. Transition metal retention andelution is dependant upon the equilibrium between free and complexedmetal. This equilibrium can be shifted by changing the pH and organicacid concentration. Generally, metal retention is inversely related to themetal-organic acid binding strength, concentration of organic acid, andeluent pH. Tartrate is the organic acid of choice but others can be usedwith an expected change in selectivity.

The use of reverse phase columns with OS gives a secondary ioninteraction separation mechanism commonly referred to as ion pairingreverse phase. In this mechanism the use of organic solvent inverselyeffects retention. For dynamically coated transition metal separations, asmall addition of acetonitrile improves column efficiency and resolutionwith a net decrease in retention.

Using these 4 retention/separation variables, the above transition metalscan be resolved on a Waters Delta-Pak C18 column ( 4.gmm x 15cm,100°A, 5tJ.particle, endcapped) using 2mM NaOS / 35mM Tartrate /pH=3.65 / 5% AcCN within 20minutes.

After the transition metals are separated they are derivatized postcolumn with PAR. PAR displaces the organic acid and forms metal-PARcomplex which has significant absorption properties between 490 and550rim. PAR is specific for transition metals; alkali and alkaline earthcations, although retained on the column, do not react with PAR norinterfere with PAR-metal formation. The response for transition metals islinear between 10ppb to 5ppm using a 100pL injection.

Several Waters UV/Visible detectors were evaluated for response at500nm. The Waters 490 detector with Xe lamp was shown to give 25%more response than the Waters 484 with deturium lamp between 500and 546 nm and 68% more respone than the Waters 440 with Hg lamp at546nm. Using the 490 detector at 520nm the detection limit using a100L injection for Cu, Zn, Ni, Co, and Mn is 10ppb and 25ppb for Fe+3,Fe+2, Pd, and Cd.

The Waters Transition Metal method has been successfully applied tohigh acid matrices (sample pH 1), high salt (up 100mM NaCIO4), highorganic matices such as fermentation brooths and dry ashed milk, andsamples that have disparate levels of transtion metals such as platingbaths.

With this method iron+3 elutes in the void where quantitation may becomprimized. Adding ascorbic acid to the sample reduces the iron+3 toiron+2, a well retained peak free of interferences, to give total ironconcentration. Iron +3 can be quantitated as the difference between totaliron and iron+2.

Neutral organics present in 18 megohm water will effect iron+3 andcopper peak shape and quantitation. These organics are concentratedfrom the eluent at the head of the column over time and show increasingdistortion of copper peak shape. The column can be washed with 50%acetonitrile / water to eliminate the problem; but it will reoccur. Placinga C18 scrubber column between the pump and the injector will removethe organics and give better long term peak shape and reproducibility.

DYNAMICALLY COATEDREVERSE PHASE COLUMN

TRANSITION METAL RETENTION

Reverse phase C18 columns are saturated with octanesulfonate to produce a column with cation exchangecharacter.

The cation capacity can be increased with increasedoctane sulfonate in the eluent.

In octane sulfonate only eluent, transition metal arestrongly retained.

J.

MM �MSC S )3 S

b

(

1

1

, !,<.......;...._._;. _ _,_ _ _

.......:.._-_,';'_"o._._.;:.

DYNAMICALLY COATEDREVERSE PHASE COLUMN

TRANSITION METAL ELUTION

Transition metals form neutral and/or anionic complexeswith organic acids such as tartrate. These complexeshave no affinity for the columnsurface.

Transition metal selectivity is controled by thefollowing general equilibrium,

Org Acid-+ TM'_TM(OrgAcid)

The stronger the complex the earlier the the elution.

Transition Metal retention is controled by the following-Concentration ofOrganic Acid-pH of the Eiuent-Concentration of Organic Solvent

• ¢n

Tar- ,,"_',,/__ _ ,,_,_ _ MI I

M(Tar)M(Tar)

MM+ M+

, !<<<

•...el

I)YNAMIC COATED REVERSE PilASE COI.,UMNSTRANSITION METAL ANAI,¥SIS

l_l_'li'l_C'l' ()1_'ORGANIC ACII) (_()N(?I_,N'I'IIA'I'II)N

Cad.,.,,: Della i'ak CI8I_.l.e.l: 2raM NaOS / 5% AceltJnilrile

Tarlrnle / pll 3.4 and 3.')

,1() r_l. 20

I%llnI'i' i2

,'10 15

"_ _ I,'l .t.,| I"e,2 I,I' 3.9( :ll I ',1

10 5 Zoo

I'hI'b

0 • i • I ,_ I i • i O- • " v u ifl :9() :l IJ ,111 !; (I 6 I1 II ? I) :111 ,111 i.;II t_il

aliM "l'inu'lriill: liil%l 'l'iirlllile

I)YNAMI(: COA'I'ED I{EVEI{SI_ I'IIASE COLUMNSTRANSITION METAl, ANAI_YSIS

EFFECT (IF pll

I)YNAMI{; (;OATF, I) i{EVEI{SE I'IIASE (_(}I.IJMNS'I'RANSITION METAl, ANALYSIS

I_l_'l_'E(_'l'( )1_'ACE'I'ONITRll ,E (_.()NCI_NTIt A'l'l( )N

3O M||

(_'.luam0: Della Pal( (;18

"_ Eluenl:" 2ram Na()S/Acelmlilrile

_ 50ram Tarlrale / pll 3.420

(;oI('

IO Zn n_ _.

Cu_" ' =1 O

0 1 2 3 4 5 6

% Acehmilrile

OPTIMIZEDTRANSITION METAL PROFILE

USING DELTA PAK C18

_;I, _W...q,I_!!!U._I;!,I, l_i!, !

I_,I,, (_lj - t).5 Iq,_,I'I_ _ II) IJl'"

Z,, ':" /.,I ---il.'.;I_l,ll,

"_" 111 Hi --- I.II I_l_llt

uJ ILI'b Ill _ (;(J = U.;; I)1,,,

If, io I'(I -- I l) I_I_'_'r_ '" I i: --I III_|)ItI

0 Ihlm (,,d,,) IG

Mi;lhod: NI-301

(._ohllllll: WIllCl_ I)clla l)nk'" (-;IR, loo °Al_,luenl: 2 him N=i('.)S , 35 hiM 'l'arl=lric nci,I, 5% A(;N (pII .3.6.5)l)elecllon: I IVIVI._ :,I 5211rim. l_.sl-c,_himvwdc,iv;ili/.:ili,,i willl I'AR

PHOTO DIODE ARRAY ANALYSISOF PAR-METAL COMPLEX

Shown below is the Waters 991 Photo Diode Array analysis ofthe transition metal profile using the Delta Pak conditions.Note that PAR-transition metal complex exhibits a broadabsorption curve between 490 and 550nm; uncomplexed PARabsorbs in the low 400nm region.

The PAR-transition metal complex is linear between thedetection limit and 5ppm; over 5ppm the absorbance exceeds2AU.

The detection limit using a Waters 490 UV/Vis detector at520nm of the highly responsive metals copper, zinc, nickel,cobalt, and manganese is 10ppb with a 100_L injection. Thedetection limit for iron+3, iron+2, lead, and cadmium is 25ppb.

These detection limits can be improved upon by monitoringeach metal at its absorption maxima.

llli II I II I i

k._, Waters 991 Three dimensional plot *** [ TNStd72398 ] 97-23-1990 15._0%in ,.. .... ........................... o .................. . ..... ,.,,°o°o.. o .......................

p o Q o _

.et$

501_WiveI encJt h4J0 -- 6130nm

(2 nm)

S_mp. time :23msec,4

B_seIine = off608 ', ', , '. ', ",

" i ie 5 o 15Time 8---20min(Isec)

.... •_ ! ( _ l ! "_

!*** Waters 991 SPectrum analgsis *** [ THStd7239e ] 07-23-1990 15:|.IS

g

:J

-r

II

DN_ .

.............. o............. - .......................... •............ - .......................... ; .......................... o .............

;'_ i ! :: ! i :: 5

_-I_"q-'-'-"-I ' " "I " r'l-'-' "i" ' ' i'_ 'q_'-'-'-I " ' ' I ' * "I**' _i' ' " I" " "1" ' "I ' ' " I " '' I' " "I ' " ' I'_ i '1' ' °' F |_" O 2 4 6 8 10 12 14 16 18 20 |

SamP. time =23msec*4 Time O---213min(l_ec)

Baseline = o(! Y-__calestep =.OSAU .01 (AU)

*._* Waters 991 SPectrum analysis ._**.... L_J__,I _..l, L_, ....__ I , I, I_.____ I , I,__l, , I,.J ,_l_.J_____ 87-23-1990

' 15:_is'__'/;i'i"/_'_i....._lii

- 23,_,..f- "_ Y - scale i

/ " _l:: _ 399 v

,--. J" ._

:::) : _ ,_ . . '\ ...............:t NicKel,,--_.:.

I ......... I,-4 ....................j:..._) " .N_..-1 F,,|tb. I

° ;: ii:l! i\ .....M._......2.16.II.........................::...:,..............................\\,,.......................................,.....' ,, ""k..\_

.!.,,",_ _,',,,.-.,_.,.."" \.'" "--.---L'-,,(__,, .,,.,.- "\

l _:__ --.; I'11_1'

;i,,_ \ .._ -'>'-_.-:__..............................:...../ l:"1[("T-' -I...._ I.....' "-T _I_, i T , ......... , .... "I"" "I"...... T _-

I ' I' I i I ...... I Ill I -i II i . ....................................

400 450 5OO 550 6OeI:lavelen_r, 400 --- __e_flnm (2nn_)

e-_, Waters 991 SPectrum analysis _ e?-23-199eo .... , I.___L____L_,I , , I , I____hj__l , I, I , L_,__I., L_.,__1 I, L, !, I,, ' 15:5

•°..o...°..)......e......).°.),.i.. i.

C _,_v_-f,_ SamP1in9 t!......--,-- 23 ms

-,I" "%. ,, ..... _H.,..io,)i1. )**.I.,)),o)e° ,.

' " ]e,< "-' Y - sca.,' D,',-- "_.

)"-" "--{'. -'-:_-_'-..

..... I if' i _.l_l. ,,°°.O.°)+.)I.,).OGI.°..O_.)Q._I.. )°.

° 7Ji <k !, .................... ; .................. '.'' ......... ;............ : .............................................

,,,,,,,""'" TMStd7/_ , , ¢ • ,i •

• ,,' / : 'l\ :

• :'1 ::::I:,"o-,,'3 I\ ._..o._......l._..P.Im(3;) ' )' ! .__.----.___. 1% .• ,, / .- . "-.-. I ), .• ,. .-- . .. , . Retention t,_ . ,,/ ,.- • .,. _\ . ,_I_i.......................'...........,'..I.,.._.......'.............'-.,..',..'t....."............................

), ,,, - _. ,..._,,. _ 2. 1.5,4I ; <" "",_ ',

," / "'t \ Pb-1 2.9

:'_ " ,,' _ ;;i ',,".,: .s j ?.,.. .., ,,,., ,' -" : ', '--:", t:_'z" 2 12.24

et / ! / : ;,N"' ",,,...............................i.,...../....:.......................,,:,_-..,._.....................

," s' ', \\,, t- ".,;\ '-.'-"----._.__• i ,,,_\ ,,._.

I. . %"%. _ ,'.-i o ) °oi,.. • '% • II....'....r-"........'--r-'-r _T' .....) ' i_r_"_r "'+-r-* ......................................

400 450 51]I] 550 G_JOWavelengtl_ 400 --- FS.Anm (2nm)

Translllon MOral PAR Respo.se Llnearlty

, 30 490 at 520nmvt

o Cu2+

Zn.+

_i _oNIZU.

Fe2

10

_,,PbZ,CdZ

"" WalorBO.l 0.5 !.0 lO,gf2

ppm

ANALYSIS OF IRON+3

Using this method iron +3 elutes in the void, a region whereother strongly chelated metals may elute. Iron+3 can bequantitated as the difference between total iron and iron+2which is a well retained peak free of interferences.

The sample is analyzed for iron+2 concentration. Then 50t_Lper 10 mls of 5% ascorbic acid is added to the sample; thisreduces the iron+3 to iron+2. The sample is injected todetermine total iron.

Shown below is the typical transition metal standard beforeand after addition of ascorbic acid. Note that ascorbic aciddoes effect the copper peak shape, however, subsequentinjections show that copper peak shape returns.

Transition Metal Standard, No Ascorbic Acid

i +tI iIi i i i i . i l i i | i i i i i i

'"" + ,+I I_I+ t

C&

*0 I I

rlt nu t.e_.

Transition Metal Standard With Ascorbic Acid Addition

; ....... , ....... • ........ ,, -.,:_, ,m',/

t;'-'0

FgLt_?0 P_

C=L

30

60

_.-_ - " ' 5 ' 10 .... 15 -20i'linute5

_0

Transition Metal Standard _ Ascorbic AcidK3E'xT I _3Ec._'T_o_

llilj'l

t20

lee _ca,

::',n _._s _ Fe,="

40 i

'_J '5 ' 10 ' 15 20

Minute5

EFFECT OF ORGANICS IN 18 MEGOHM WATER

The transition metal method employs the use of C18reverse phase columns. With the Delta Pak eluent, anyneutral organics present in the D! water will tend toconcentrate at the head of the column. The organicsusually found in 18 megohm water are polar and as suchcan act as potential chelation sites for the metals.Copper is the only metal that is effected by theseorganics; copper peak shape will distort and finallydisappear as the concentration of the organics on thehead of the column increases. This usually occurs within1 liter of eluent passed through the column.

This problem can be eliminated by washing the columnwith 50/50 acetonitrile/water for 15 minutes followed

by 30 minutes of equilibration with fresh eluent. Ameans of preventing this problem is to install a C18scrubber column, a used Delta Pak C18, between thepump and the injector to remove the organics from theeluent prior to injection of transition metals.

Experience has shown the quality of the 18 megohmwater is critical in customer satisfaction. Normally thisorganics problem is not evident during/socraticreverse phase HPLC or ion chromatography so customerdoes not believe the problem is with their water.Washing of the column with acetonitrile followed byequilibration with organic free eluent, or by installingthe scrubber column, is the best means to demonstratethe problem is with their 18 megohm water.

Shown below are chromatograms showing the effect oforganics in the water, and the transition metal profileafter acetonitrile wash, and a linear acetonitrilegradient analysis of the contaminated column showingthe organics concentrated on the column.

Transition Metal Profile of an ._

Organics Contaminated Column

i i i i i •

::;-j_.) -

"'J"/ I[,1

//

• C¢, il

NkP :! ,

'_0 " H .t

_ pu i' t iI ,

"_,:,o ] ;" i_ I ca Fe"

•; --: 1._:j " 5 _8"11ITUt.e-

Transition Metal ProfileAfter Acetonitrile Wash

i

r-

-"Oe " C_ [Iff!Jol

,-"5¢, "_ t'l _"/

- iI Z_ Coi J

ae,-, 7

tso [ ., p_

J - i •' 0 5 18 1,5 _.:'8

Mi t'=ut.e_.

Organics Profile of Contaminated ColumnUsing a Linear Acetonitrile Gradient and

Detection at 210nm

............. [00%

ft'|"¢_ i /'%

150

5o J

- "_.- - : . :i " ;30 4A _SA '6r_ e=" 1 =:1 _ _.t

I'1t raut.@_

I ransltion Metal Prolile o! Uruoe HaUl salt

HIGH SALT MATRICES SAMPLE: lob Crude NcAI in DI WaRESULTS:

Zn .747 ppm

.0! AIJ _ Fo .025 ppmMn .O79 ppm

TRANSITION METALSr'-

IN NaCIO4 o ® :_

l_ IOOmM NaCI()4

Blank

25 minutes Waters

I-f

__ _. ..._ ,__,.__._______._: .___ Transition Metal Profile of Soil ExtractSAMPLE: 4 grams Soil Suspended In 100 mls 3raM Sr (NC-, ................ . ...... ,-, ...................................... ,.............. RESULTS:

,1t ,,, t Zn .078 ppm

Zn 100raM NaCI()4 Cu = 25 ppb Co .00I ppm

('u (!o Standards I'b = 50 ppb / ' Mn .026 ppmMn i = 50 ppb

Ni [I I] (:o = 25ppb J

I'b Ire = 50ppb " ® c

; :-. a _. _ Io !_:" t4 t6 20 minutes Walers

HIGH-ACID-MATRIcES I _ 'i

TRANSITION ME'I'AlJS

IN HBr Transition Metal Profile

HNO 3 Digested WastewaterSAMPLE: HNO30igesled WastewalerRESULTS:

Cu = 12 ppl) C. .0o8 ppm

1-- tNi = 15 ppl) .002 AU Zn .024 ppmNi .030 ppmFe = 46ppb Co .003ppm

.147_ cdr_ + Mn .034 ppm

¢_ • I_ ...... --'--"_ a;z o

,,.................................._ Jo 15 " ' " ' _d ...... " 30 minutes WatersHi,,,.,,e_-- 10,978

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"_ :'"_'- :.. _, ,,DISPARATE METAL CONCENTRATION "_;_;_:

..._ .... :_,,-•.-.•

Trace Metals in Acid Copper Plating Bath Trace Metals in a Nickel Plating Bath

c SAMPLE: 1:1000 Dilution of Plating Balh SAMPLE: 1:1000 Dilullon of Plating BAt,,N in 5% HNO3 in 5% HNO3

:. = tl = II RESULTS: RESULTS:I / < II o /dI Cu - 1 Cu .oo3DpmPb .028 ppmJ.o_ II "]1 z. 3.42 ppm .02Au -_J• II /I Ni .97 ppm o0_3 _pm

II /I -_ co .oo3 ppmII /h Fe 2.13 ppm Co _01"3ppm

kin .003 ppm

..... 0 ,e

25 minutes 25 minutes

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U Oo °25 minutes 25 minutes

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HIGH ORGANIC MATRICES0

Transition Metal Prolile

Transilion Metal Profile Ashed Milk Resuspended In HNO3

Clarilled rlecolnbhlalll DNA SAMPLE: lf_ mls AshedMilk Resuspended In .5% IINO::

Fermentation Broth RESULTS:Pb .305 ppm

SAMPLE: 1:20 In Eluent

RESULTS: '<=

cu 23 ppm I_Zn 3.6 I)pm

.OIAU Ni 1.1 ppm iCo .2 ppm (_

c_ : Fo t.6 ppmMn 1.0 ppm

o..... Wmlers - -

-- 10,902 30 m|nules Writers............. 10,980