Reverse Phase CHR and PH Control

 

TB 99-06

Importance of Controlling Mobile Phase pH in Reversed Phase HPLC By Aimee N. Heyrman and Richard A. Henry INTRODUCTION The importa nc e of co ntroll ntrolling ing mob ile ile p h as e p H w h e n an al yz i n g i o n i z ab l e c o m p o u n d s b y re v e rs e d p h as e ( R P ) HPLC is often recognized a nd ea sily sily understood , how ever iitt is is often eq ually ually important to c ontrol pH whe n wo rki rking w ith field samples of non-ionizable compounds due to the pr pres es ence o f ioni ioniaa ble impuritites.

Figure 1. Buffer Capacity

% Buffer Capacity

% Ionized

SELECTING SELEC TING THE THE RIGHT BUFFER BUFFER A pa rtia rtia l lis t of c ommo n buffers buffers a nd the ir corresponding pH values is shown in Ta b le 1 1. P erhaps erhaps the the most common HPLC b uffer uffer iiss so me form of phosphoricc a c id. A de finiti ri finition on of buffer st reng th is g iven in Fi Fig ur uree 1, w here a plot of how  co n ju g ate fo rm s o f p h o sp h o ric acid cha nge with pH. Note that buf buffer fer ca pacity (the ability to resist pH change w hen a s a mple is is introd introd uced a t a d iffer ffer-ent p H) is only 100% a t the pK va lue lue o f the ac id or ba se. At pH 4 4,, phosphate is a poor buf buffer fer a nd w ould ould c hange rapi rapidly dly toward one of its pK a   values if a more ac idic dic or ba sic s a mple mple w er eree intr introduced oduced . As a rule, rule, o ne s hould w ork withi within n ± 1 pH unitt of the buffer pKa  value for uni for good pH co ntrol ntrol of the mobile mobile phas e. Adeq uate buffer concentrations for HPLC tend to be in the 10-100 10-100 mill millimolar level dep end ing on the s ize a nd na ture ture of the s a mpl mple, e, as we ll a s the column column pac king ma teri teria l. P has es that conta in pola pola r groups groups such a s AQUAS QUAS IL C18 and P RI RIS S M RP, RP, a re often mo re co mpa tible tible w ith d ilute b uff uffers ers tha n trad iti tiona ona l alk alkyl yl pac ki kings ngs .

used . If control a t pH 4-5 4-5 is d es ired, a n organic acid buffer such as acetate or citraa te sho uld citr uld be co nsidered in pla pla ce of phosphate.

When control at a lower pH (2-3) is desir sired, ed, phos phate, o r stronger stronger orga orga nic nic a cids suc h a s TFA or ac eti eticc a cid w hen volatility is of concern, are commonly

Table 1. Properties of Common Buffers C o m m o n B u ffe rs

p Ka

Useful pH Range

P ho s p ha te

p K1 pK 2 pK 3

2 7 .. 1 2 1 2. 3

1 6 .. 1 2 --3 8 .. 1 2 1 1 . 3 -1 3 . 3

C it ra t e

p K1 pK 2 pK 3

3. 1 4. 7 5. 4

2 . 1 -4 . 1 3 . 7 -5 . 7 4 . 4 -6 . 4

Fo rm a t e

3. 8

2. 8-4 . 8

Ac e t a t e

4. 8

3 . 8-5 . 8

Tris

8. 3

7 . 3 -9 . 3

Am m o n ia

9. 2

8 . 2 -1 0. 2

B o ra t e

9. 2

8 . 2 -1 0 . 2

D ie t h y la m in e

10. 5

9 . 5 -1 1 . 5

 

WHEN IS pH CONTROL NECESSARY NECESSARY FOR STRONGLY STRONGLY IONIZABLE COMPOUND C OMPOUNDS? S? S a mples co nta ining ning ioni ioniza za ble compo unds a re strongly infl influenced uenced by pH of the mobile mobile pha se a s ill illustrate d b y the sepa rations of sorbic a nd be nzoic ac ids sho w n in in F Fiigure 2. 2. Whi hille orga ni nicc a cids a re typica lly sep a rated under iion on suppress ion co nditi nditions ons w here here the pH is a djuste djuste d t o 2 o r 3 (Fi (Figure gure 2A) 2A), this s a mple req req uir uired a pH of 7 (Fi (Figure gure 2B) due to the tende ncy of the s a mple ma trix trix to pr prec ec ipi pita ta te a t low low er pH and higher orga ni nicc c onc entrations. Low pH dec rea se s the so lubili ubility of or orga ga nic nic a cids in w a ter and req ui uirres the us e of a higher orga ni nicc perce nta ge in the mob ile pha se for prac ti tica ca l eluti elution on under RP c onditi onditions ons . For ac ids , the rretenti etention on time time dec rea ses a s the pH of the mobil mobile phas e is is in increas creas ed . G rea ter cha rge c a n be thought thought of as a n extreme ca se of polari polarity. ty. At pH’s pH’s a bo ve the ana lyte’s pKa , the acidic analyte carries a negative charge and behaves as an extremelyy pola extremel pola r molecul molecule. e. In or orde de r to ac hi hieve eve ad eq uate retenti retention, on, the mobile mobile phas e should be hi highly ghly a q ueous. Be low its pK a , the ac idic ana lyte is is neutral a nd much more h hydrophob ydrophob ic. Under iion on suppresion RP retention ca n be more eas ily ac hi hieved eved a nd a nalysi nalysiss times c an b e longer longer.. When the acid standards were introduced to unbuffered, neutral mobile phase of 10% methanol (Figure 2C), poor peak sha pe resulted. resulted. Thi hiss result result ca n be traced to a mi misma sma tch ca used b y the ac idic nature nature of the sa mple mple a nd zero buf buffer fer strength in the neutr neutraa l mob ile pha se . The sa mple ther therefore efore experi experience ence s a pH grad ient during during the first first pa rt of the se pa ration, which usua lly ca uses ionizab le co mpounds to exhibit exhibit broa broa d pea k sha pe a nd poo r rretention etention reproduc reproduc ibili bility. As s how n in in Fi Figure gure 2B, ad diti dition of a phospha te buff buffer er at pH 7 eli elimi minated nated the broad taili tailing pea ks a nd c rea ted rugged cond iti tions ons s uitab uitab le for succes sful a ss a y. Bec a use the a ss a y of sorbic and benzoi benzoicc a cids wa s rel relaa ted to a food product product co ntaini ntaining pr protei otein, n, the wide wide pores a nd neutral pH prevented prevented prec prec ipita pita ti tion on a nd prolong prolong ed c olumn olumn lliife. Ess entiall entially, the s olutes olutes a re insta insta ntl ntlyy ionized ionized a nd the w ea k a cids a re se pa rated in th their eir a ni nioni onicc forms. Thi hiss pH grad ient eff effec ec t is is diffi difficult to co ntr ntrol ol a nd ca n almost g uara ntee problems w ith reproduc ibil biliity.

CONCLUSION:While it is not always strictly necessary to operate under buffered conditions, one should recognize that poor pea k sha pe a nd va ri riaa ble retention retention ca n res res ult ult w hen the sa mple pH diff differs ers s ignifi gnifica ntly ntly fr from om the pH of the nonbuffer buff ered ed mobil mobilee pha se a nd w hen ioni ioniza za ble compounds a re present in in the sa mple. mple.

Figure 2. Effect of pH Control on Separation of Ionizable Compounds  A  A:: pH 3.5, buffered

B: pH 7.0, buffered

C: pH 7.0, not buffered

Sample: 1. B enzoi enzoicc A Aci cid d 2. Sorbi Sorbicc A Aci cid d 2

2

2

α1,2=1.1 1

α1,2=1.5 1 1 721-009

721-010 0

10

20

BioBas BioBasic ic 18, 5µm, 150x4.6mm Part No: 155-7 5-721 21 El Eluent: uent: 20% MeOH/ MeOH/80% 80% 0.05M 0.05M KH2P O 4, pH 3.5 F low: low: 1. 0 m L L/ /m in in D e t e c to to r : U V @ 23 23 5

MIN

0

2

4

BioBas BioBasic ic 18, 5µm, 150x4.6mm Part No: 155-7 5-721 21 El Eluent: uent: 10% MeOH/ MeOH/90% 90% KH KH 2P O 4, pH 7 F llow: ow: 1. 0 m L L/ /m iin n D e t e c to to r : U V @ 23 23 5

2

6

MIN

321-057

0

2

4

BioBasic ic 18, 5µm, 150x4.6mm BioBas Part No: 155-7 5-721 21 Eluent: Eluent: 10% MeOH/90% H2O F low: low: 1. 0 m L L/ /m in in D e t e c to to r : U V @ 2 23 35

6 MIN

 

WHEN IS pH CONTROL NECESSARY FOR NON-IONIZABLE SAMPLES? DELTAB OND AK w a s eng ineered for the as sa y of aldehyde s a nd ketones prese nt in DELT in auto emiss ions . S ince then, other groups ha ve employed DELT DELTAB OND AK for for the as sa y of aldehyd es a nd ketones in amb ient a ir sa mples a nd s a mples from from other sites w here aldehyd e a nd ketone polluti pollution on is prese nt. Traditiona raditiona lly, the sa mples a re fi first rst d eriva eriva ti tized zed w ith DNPH for ea sy dete ction by UV-V UV-Viis, then a ss a yed w ith mobile mobile pha se c ons isting of H2O/ O/A AC N. The DNP DNP H-deri H-deriva va tized a ldehyd es a nd keketones are not ionizable and therefore do not require a buffered mobile phase. Auto emiss ions ge nerally nerally a re free of ionizab ionizab le c ompo unds; how ever, ever, recently the usua l method w a s used w ith DELT DELTAB OND AK for for the a na lys is of a ldehyd es a nd ketones in a mbient a ir sa mples s ur urrrounding a fac ility w hich hich m a nufac tures tures nylon nylon ((Fi Figure gure 3A) 3A). Although the sa me method ha d be en used s ucc es sfull sfully for this this a na lys is a t other ma nufac tur turiing fac iliti ties es , sudd enly enly a la rge g host pea k wa s o bs erved erved tha t iinter nterfer fered ed w ith the qua nti ntitation tation of formal formaldehyde. dehyde. The ghos t pea k ha ha d poo r peak shape and wa s not pr present esent in in bl blaa nk runs runs or stand ards . Although the the pea k sha pe wa s a lwa ys b roa d, the retenti retention on time time varied varied co lumnumn-toto- column and lot-to-lot, ot-to-lot, indica indica ti ting ng tha t the g hos t pea k wa s interac ti ting ng s tr trong ong ly w ith the residua residua l sila sila nol gr groups oups on the surface of the silica. The H 2O co mponent of the mobile mobile pha se wa s repl eplaa ced with a b uf uffer fer (KH2P O 4  pH 2.5 with H 3P O 4) (Figure 3B ). The la rge spurious spuri ous pe a k shifted to the solvent ffrront, ma ki king ng a cc ura ura te q uantitation of the the formaldehyde pe a k pos sible. The rretention etention ti times mes of the aldehydes and ketones ketones were unaffected unaffected sin since ce they are not ioni ioniza za ble and theref therefor oree a re unaffected b y cha nges in pH. pH . The gho st pe a k is mo st likel likelyy a a mine mine co mpound tha t has bee n pa rti rtiaa lly deriva deriva ti tized zed w ith DN DNP P H. A diamine is is us ed in the ma nufac tur turiing proces s of nylon, which which c ould ould b e the so urce urce o f the spurious spurious pe a k. The DN DNP P H deri derivat vat iza ti tion on of one of the a min mino o g roups rende rs the o ther amino group in inaa ctive, res res ulti ulting ng in a pa rti rtiaa lly d eri eriva va ti tized zed co mpound tha t is s ti tilll ionizab ionizab le a nd co nseq uently uently doe s not b eha ve we ll under the non-buf non-buffer fered ed c hr hroma oma tog raphic cond iti tions ons . Although unbuffered, unbuffered, the curr current ent mob ile pha se is a pproxi pproxima ma tely pH 7 7,, resulti resulting ng in parti partiaa l ioniza oniza ti tion. on. Mobil Mobilee pha se pH will will cha nge s lightly as the percent organic in in the mobil mobilee pha se c hang es , which in in turn turn affects the percent iionization onization of the the gho st pea k. The rretention etention mecha nism nism o f this this pe a k iiss proba bly due iin n pa rt to iion on exc ha nge effect s w ith the res res idua l sil silaa nol groups, w hich hich c a n vary fr from om lot-tolot-tolot a nd even co lumnumn-to-column. to-column. Thi hiss effect c a n be elimi elimina na ted b y bufferi buffering the mob ile pha se a t ab out pH 2.5. 2.5. Unde r thes e conditions the amino compound will be extremely ionized, move to the solvent front, and behave consistently and the sil silaa nol nolss will will n not ot d iss ociate a nd impart nega ti tive ve c harge to the pa cking. cking.

FIGURE 3. Effect of pH Control on Separation of  Non-Ionizable Compou C ompounds nds CONCLUSION:  M o b i l e p h a s e p H should be controlled when assaying non-ionizab non-i onizab le o r neutr neutraa l ana lytes in the presenc presenc e of ioniza ioniza ble conta minants minants or impurities. mpurities. Ioniza ble co mpo unds a re easily recognized by their inconsistent

B: 0.05M KH2PO4, pH 2.5/ACN

A: H2O/ACN

Sample-DNPH derivatized ambient air samples : 1 . F o rm rm a ld ld e h y d e 2. Ac et a l d eh yd e   3. Acetone   4. Acrolein

run-run and sample-sample behavior under non b uff uffer er co nditi nditions ons .

G rraa d ie n t :

4 1

12 15 17 19

Impurity

Impurity

T %A %B 0 65 35 8 65 35 35 35 65 65

65 65 35 35

T= Time (min.) 2 3 1 2

34 209-008

209-007

0

2

4

6

8

10

12

DELTABOND OND AK, 5µm, 150x4.6mm DELTAB Part No: 155-2 5-209 09 E llu u e nt nt : A H20 B: ACN F llow: ow: 1. 5 m L L/ /m in in

14

16

1 8 M IN

0

2

4

6

3

10

12

14

DELTABOND AK , 5µm, 150x4.6mm DELTAB OND AK, Part No: 155-2 5-209 09 Eluent: Eluent: A: 0.05M KH2PO4 in H2O, pH 2.5 B : AC N Flow: Flow: 1.5 m L/min D et ec t or: UV @ 3 365 65

D e t e c to to r : U V @ 36 36 5

8

16

1 8 M IN

 

IMPORTANCE OF CAREFUL pH CONTROL? S mall cha nges in the mobile mobile pha se pH ca n also ha ve a drama ti ticc eff effect ect o n the selecti selectivi vity ty of w ea kly iioni oniza za ble compounds. A sa mple of 7 comm on a nti ntiiinfla nfla mma tory drugs w a s s epa rated a t pH 2.1 (Fi (Figure gure 4A) 4A) a nd pH 2.5 (Fi (Figure gure 4B). 4B). Although 6 of the 7 a nalytes beha ved ve ry simil simila rl rlyy under b oth c onditions onditions , Dif Diflluni unisa sa l el eluted uted a pproxima pproxima tely 1 minute minute ea rl rliier at pH 2.5 than a t pH 2.1, iindica ndica ting ting tha t it it is is mo re ionized ionized a t the higher higher pH. Thi hiss b eha vi vior or indica indica tes the presenc e of a ca rboxylic rboxylic a cid g roup in the molecule that w a s se nsiti nsitivi viee to pH in in thi thiss ra nge

uld a lw a ys b e employed w hen wo rk rkiing w ith mil mildly iionizab onizab le co mpounds to ens ur uree CONCLUSION: Adeq uate pH c ontrol sho uld maximum run-run reproducibility.

O C OH F

OH

F Diflunisal

FIGURE 4. Effect of Small Changes in pH on the Separation of Mildly Ionizable Compounds

A: pH 2.1 23

Sample: 1. Uracil 2. To lme tin 3. Naproxin 4. Fenoprofen 5. Diflunisal 6. Indometacin 7. Ibuprofen

B: pH 2.5

2 3

1 7

1

4

7

5

4

5 6

6

701-085 0

701-086

6 MIN

0

BETAS BETASIL IL C18, 5µm, 50x4.6mm Part No.: 155-701 El Eluent: uent: 50% ACN/50% 25mM H3P O 4, pH=2.1 Flow: Flow: 0.8 mL/min min D et ec tor: tor: UV @ 220 220 nm nm

6 MIN

BETASIL IL C18, 5µm, 50x4.6mm BETAS Part No.: 155-701   Eluent: 50% ACN/50% 25mM KH2P O 4, pH=2.5 Fl Flow: ow: 0.8 mL/min D et ec t or: or: UV @ 2 220 20 n nm m

4

 

USE OF ACID MODIFIERS MODIFIERS TO ADJ UST pH It is is a lso co mmon to employ strong or we a k ac ids a lone to co ntr ntrol ol pH at low va lues (Fi (Figure gure 6) 6),, a s s how n iin n Ta ble 2, for co mmonly used trifl trifluoroac uoroac etic (T (TFA) a nd a ce ti ticc (HAC) HAC) ac ids . For a more thorough treatme nt of this top ic plea plea se se e referreference 2. Equa ti tions ons used to c a lcula cula te a ppr pproxi oximate mate pH values values are s hown in Figur Figuree 5 for strong (nearl (nearlyy d iss ociated) and wea k acids, where C a  is the c onc entration of the a cid in mol/ mol/L and Ka  i  iss the a cid-dissoc cid-dissoc iation consta nt. As s how n in in the ca se of TF TFA, ca lculated values c a n dif differ fer si signifi gnifica ca ntly ntly fr from om mea sured va lues w hen the a cid ha s properti properties es betw een that of a w ea k a nd strong ac id. Equa ti tion on 2 iiss a more ri rigorous estimation estimation of pH than eq uati uation on 4 and offer offerss a better a pproxi pproxima ma tion tion of pH for modera te a cids suc h a s T TF FA. When T TF FA a nd HAC HAC a re use d, this method of pH control do es not provide provide a buffer buffered ed m ob ile pha se a nd ma y not be a s effective for a ll types of sa mples, es pec ia lly ba sic ones . How ever, ever, iitt has be co me popular ffor or ad justing the pH of mi milldly ionizab le c ompo unds s uch a s pe pti ptides des a nd proteins. proteins. As Figure Figure 6 il illustra tes , TF TFA ca n be us ed to not only control mob ile pha se pH but a lso the s electi electivi vity ty a s w ell ell.. An order of mag ni nitude tude c hang e in co nce ntr ntraa ti tion on of TF TFA res ults ults in a s ignifi gnifica nt cha nge in pH a nd a dra ma tica lly lly d ifferent fferent c hroma tog ram . At 0.1% TF TFA (pH 2.0) (Fi Figure gure 6A 6A)), A Angiote ngiote nsins II and III coe lute a nd a t 0.01% TFA (pH 2.4) (Fi Figure gure 6B), they a re ba s eli eline ne reso lved . While hile so me of the c ha ng e ca n be a ttribute ttribute d to ioniza tion differ difference ence s a t the tw o pH va lues, TFA a lso ha s uni uniqq ue properti properties es w hi hich ch m a y resul resultt fr from om its reported a bili bility to form strong ionpairs with positively charged species.

Table 2. Properties of Acid Modifiers Mo d if ie r (v/v)

C o n c e n t ra t io n (Mo le s /L)

Me a s u re d pH

C a lc u la t e d p H (1)

C a lc u la t e d pH (2)

C o ns ta nt Ka

B u ffe r pK a

1 . 3 5 x 10 -2

2. 04

1. 09

1. 89

0. 50

0 . 30

6 . 75 x 10

-3

2. 20

1. 23

2. 18

0. 50

0 . 30

0 . 0 1 % TFA

1 . 35 x 10

-3

2. 44

1. 58

2. 87

0. 50

0 . 30

1 . 0% H AC

1 . 75 x 1 0 -1

3. 01

2. 75

2. 74

1 . 8 5x 1 0 -5

4.74

0 . 1 % TFA 0 . 0 5 % TFA

1. pH ca lculated lculated using using Eq uation 4 2. pH ca lculated lculated using using Eq uation 2

Figure 6. Effect TFA Concentration on Separation of   Mildly Ionizable Compounds

Figure 5 Weak Acids

− K a ±

2

K a 

+ 4 K a C a

(1)

[ H + ] =

(2)

= − log   pH  ~

K a 



2

A: 0.1% TFA (v/v) pH 2.0

2

 − K a ± 

2

+ 4 K a C a   

B: 0.01% 0.01% TFA (v (v//v) pH 2.4 Sample: 1. Angiotensin III   2. Ang ioten sin II 3. Angi ngiotensi otensin n I

3

1, 2

(3)

[ H   ] =

(4)

=  pH  ~

+

3 2

K    a C a

 − log  (K a C a )  2 

1

Strong Acids (5)

 H 

+

  = C  A 321-051

(6)

 pH  =  − log C a

0

5

10

PRISM RP, 5µm, 150x4.6mm Part No.: 155-321   Elu Eluent: ent: B A:: 0. 0.01% T H2O 01% TFA TFA FA in iin n ACN 10% →50% B in 20 min. Fl Flow: ow: 1.0 mL/min D et ec t or: or: UV @ 220 220

5

15

321-053

MIN 0

5

10

PRISM RP, 5µm, 150x4.6mm Part No.: 155-321  

2 Eluent: A: 0.01% TFA TFA in O B: 0.01% in H ACN 10% →50% B in 20 min. Flow: Flow: 1.0 mL/min D et ec t or: or: UV @ 220 220

15

MIN

 

Figure 7. Tryptic Tryptic Digest Digest of β−Lactoglobulin on BetaBasic 18

Fi Figure gure 7 is is a nother exa mple of the importance of the ac id c oncentrati oncentration. on. Fi Figgure 7A shows a separation of a tryptic digest of β-Lactoglobulin with 0.01% TFA w hile hile Fig Fig ure ure 7B s how s the s a me se pa rat ion w ithout TFA prese nt in the mobilee phase. In tthi mobil hiss ca se ther theree wa s dram a ti ticc loss of retention retention and s electiv electiv-ity for a ll of the peptide peptide frag ments . A trac e a mount of ac id is usua lly required required to ma intain adeq uate pH c ontr ontrol ol and impr prove ove the sepa ration. G enerall enerallyy the llow ow est concentration possible should be employed empl oyed a s long a s resul results ts s how rugged ness a nd rrepr eproduc oduc ibil biliity. ty. Lower co ncentra tions tions of buff buffers ers and a dd iti tives ves ca n reduce ma intenance req uir uirements, be more compa tible tible wi with th detecto rs, a nd improve the lifetime of columns and other system components.

B: 0.0 0.00% 0% TFA TFA pH 7

A: 0.01% TFA pH 2.4 S a mple: Trypti rypticc Dige st o f

β−Lactoglobulin

715-130 715-129 0

10

MIN

0

10

MIN

BetaBasic 18, 5µm, 150x4.6mm Part No.: 155-715 Eluen Eluent: t: A: TFA TFA in in H2O B: TFA in ACN 10% →50% B in 20 min. Fl Flow: ow: 1.0 mL/min D et ec t or: or: UV @ 220

Fi Figg ur uree 8 illu illuss trat es the e ffect o f different different a c id mod ifi fiers ers on s elect ivi vity. ty. Although the pH of the two mobile mobile pha ses varies varies by less than 1 pH unit, unit, the a cetic a cid mobile phase offers much greater selec ti tivi vity ty for the sa me pa ir of co mponents tha t a re only pa rti rtiaa lly rres es olved w ith TF TFA. Th is is is a n o th e r iin n d ica tio n th a t th e mechanism of separation, especially with organic acids, can involve specific interactions between the solute and acid, such as ion-pairing mentioned ab ove or s imply mply pH effects.

Figure 8. Effect of Acid Modifier on Selectivity

A: 0.01 0.01% % TFA pH 2.4

Sample: 1. Angiotensin III 2. Angi ngiotensi otensin n II 3. Angi ngiotensi otensin n I

23

B: 1.0% Acetic Acid pH 3.0

23

1

1

715-125 715-124

0

10

BetaB BetaBasi asic c 18, 5µm, 150x4.6mm Part No.: 155-715   Eluen t: A: 0.01 % TFA in H 2O   B : 0.01 % TFA in ACN 10% →50% B in 20 min. Flow: Flow: 1.0 mL/min D et ec t or: or: UV @ 2 220 20 n nm m

20

MIN

0

10

20

BetaBasic 18, 5µm, 150x4.6mm Part No.: 155-715   Eluent: A: 1.0% HAC in H 2O   B: 1.0% HAC in ACN 10% →50% B in 20 min. Fl Flow: ow: 1.0 mL/min min D et ec t or: or: UV @ 2 220 20 n nm m

6

MIN

The increas ed noise o bs erved in Figure Figure 8B is is c aus ed b y hi higher gher bac kgroun ground d UV ab sorbance o f 1 1.0% .0% ac etic etic ac id c ompa red to 0.01% TF TFA.

 

GUIDELINES FOR PREPARING MOBILE PHASES Bec aus e slight slight varia varia ti tions ons in pH and a cid conc entration entration can ha ve a d ra matic impac impac t on sepa ra ti tion, on, consistent certain certain techniq niq ues s hould hould b e employed w hen prepa prepa ri ring ng mob ile pha se s to ens ur uree go od reproducibi reproducibillity. As d es cri cribed bed in the liliterature3, it is generally generally a goo d idea to mea sure sure a n appropr appropriia te a mount of pur puree w a ter iinto nto a volumetr volumetriic fl flas as k with with an a cc ur uraa te a mount of sa lt or ac id. The pH of the mobile mobile pha se sho uld uld b e a djuste djuste d, if req uir uired, b y a dd ing reag ent before dil diluti uting ng to fina fina l vol volume ume a nd prior prior to blendi blending ng of a ny orga orga ni nicc s olvents olvents . For exa mple, blending blending 25% metha nol w ill raise the a ppa rent, mea sure pH of the co mbined mo bil bilee pha se by a bo ut 0.5 pH uni units. ts. Alternatively ternatively, eq uimolar uimolar so lutions utions of differ different ent ionic ionic forms of the s a me b uf uffer fer (i.e. mono a nd diba sic phospha te) ca n be b lended to reach the d esired esired pH. When developi developing ng a ru rugg gg ed me thod, it is is d es irable to se lec t a mo bile bile pha se w ith a final final pH a t llea ea st o ne pH uni unitt aw a y from from a ny a na lyte’s pK va lue to ca use ioniza ioniza ti tion on or suppress ion of the a na lytes . There is often so me gues sw ork in thi thiss b ec a use the effect of type and concentration of organic solvent on either mobile phase pH of solute pK values is not accurately known. reprod od ucibil ucibility w ith s everal ba tches of mo bile bile Duriing me thod deve lopment, it is important to monitor chroma Dur chroma tog raphic (k’, (k’, α) repr pha se , a s it ca n be d iffi fficult to co nsistently reproduce reproduce pH pr prec ec ise ly. The eq uations in F Fiigure 5 a re a g ood sta rting rting point for ca lculating the co ncentra ti tion on of a cid requir required ed to a chieve a d es ired pH for se pa ration. How ever, ever, a s Ta ble 2 sho ws , pH ca n vary signi signifi fica ca ntl ntlyy from from thos e ca lculations. It iiss therefore therefore very importa importa nt to experimenta experimenta lly de termine termine a nd report the value value of the mo bile bile pha se pH with a c a librat ed p H meter to ens ur uree reprod reprod ucible ucible results. The us e o f prepre-mi mixed xed mob ile pha se (pumpi pumping ng from a single res res ervoir) ervoir) is es se ntia ntia l to ens ur uree a cc ur uraa te a nd reproducible mobile mobile phas e co mpositi mposition. on. However, However, iitt has bec ome popul popular ar to prepar preparee a n aq ueous buff buffer er and program the instru instrument ment to blend blend organic s olvent olvent w ith aq ueous b uffer uffer ffor or grad ient elu eluti tion on or fa fa st isoc ratic method d evelopment. This his pra ctice c a n res res ult ult iin n poor a cc ur urac ac y a nd incompl incomplete ete mixing, mixing, d ependi epending ng o n sys tem ma intenance a nd c a librati bration, on, ma gni gnitude tude of d we ll vvol olume, ume, flow  flow  ra te a nd other factors. Isoc ra ti ticc methods that ha ve been d eveloped eveloped usi using ng instrumen instrumentt blending blending s hould hould b e c onfir onfirmed by premixed mixed mob ile phas es, a nd grad ient methods should should b e c ompa red b etween mo re than o ne iinstru nstrument ment when pos sibl sible. e.

SUMMARY AND RECOMENDATIONS Controlling the separation of ionizable compounds can be difficult, and careful attention must be paid to all experimental deta ils in order to a cc ompli omplish sh a rugg rugg ed metho d. S light va ri riaa ti tions ons in mob ile phas e prepa prepa ration ca n rres es ult ult iin n pH cha nges that ca n have d rama tic tic e ffec ffec ts o n selectivi selectivity, ty, ca pa city fac tor ((rretention fac tor), tor), pea k sha pe, resolution, resolution, a nd reprod reprod ucibil ucibility. OptiOptimum pH co ntrol ntrol wil willl usua lly resul resultt iin n mob ile pha se co nta ini ning ng b uff uffer er and a cid c ompo siti sitions ons tha t w ill res ist cha nge w hen the sa mple iiss introd ntrod uced a nd force ioni ioniza za ble a na lytes into predo mi minantly nantly one form (i (ionized onized or neutra neutra l) a s they enter the c olumn. olumn. G ood la boratory practice in pr prepa epa ring mo bile bile pha ses should should be foll follow ed to ens ur uree tha t results results ca n be reproduced eproduced withi within n a nd betw een la la bo ratories ratories . Whil hile iinstrument nstrument solvent bl blending ending has be co me very convenient for for fas t method d evelopment, iitt iiss bes t to eva luate p re-mixed e-mixed s olvent w henever poss ible to ensure ac curac y a nd eq uil uilibra ti tion on before co mpleting mpleting a nd publi publishshing a n H HP P LC method. This his extra s tep ca n eli elimi mina na te the poss ibili bility that iinstrument nstrument fa fa cto rs could make sepa ration res ults ults difficult for others to reproduce.

Mobile phase pH should be selected so that it is at least ± 1.5 pH units from the analyte’s pKa. This assures that the analytes are either 100% ionized or 100% non-ionized and should help control run-run reproducibility. reproducibility. At high pH, acidic compounds are ionized and are much more hydrophilic than under ion suppression conditions. These conditions should be selected when fast analysis and low retention are desired. BioBasic 18 is a good choice under high pH conditions.

REFERENCES: 1) R.C . W Wea ea st, ed., Hand bo ok of Chemistry and P hysics , ((C C RC P res s: C leveland, OH), OH), 197 1974. 4. D112D112-D114 D114.. 2) R.A. R.A. Henry Henry,, D G a ha ga n, Des ign o f V Volatil olatilee B uffer uffer Sys tems for LC LC Applica pplica ti tions ons , (Keysto (Keysto ne S cientifi cientific, c, Inc. Be llefonte, P A) 3) “S epa rations S olu oluti tions ons : Mobil Mobilee P ha se pH,” U.D. Neue, Neue, A Ameri merica ca n L Laa bo ratory ratory,, March 1999, 1999, p. 60.

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