The The Use Use Of Of A A Novel Novel Type-C Type-C Silica Silica To To Separate Separate Amino Amino Acids Acids By By Aqueous Aqueous Normal Phase (ANP) With Electrospray (ESI) Detection Normal Phase (ANP) With Electrospray (ESI) Detection

ASMS 2007 Poster WPW 377

Steven StevenM. M.Fischer; Fischer;Agilent AgilentTechnologies, Technologies,Joseph JosephJ.J.Pesek; Pesek;Department DepartmentofofChemistry, Chemistry,San SanJosé JoséState StateUniversity, University,Maria MariaT.T.Matyska; Matyska;MicroSolv MicroSolvTechnology TechnologyCorporation Corporation

Results and Discussion

ANP Column L-Alanine L-Arginine L-Aspartic acid L-Cystine

9 8

R e t e n tio n tim e (m in )

6

4

2 1

100

1.000 1

An Agilent 1100 Series HPLC system with binary pump and degasser, well plate autosampler with thermostat, thermostatted column compartment, and a Agilent 6210 MSD TOF mass spectrometer was used (Figure 2).

6

7

5 4 3 2

0.800

Retention time vs. % methanol in the mobile phase

0.600

0.400

0.200

0.000 0.0028

A A m m in inooA A ccid id LL-A -A la lannin inee

M M .W .W ..

(M (M ++H H ))

(M (M -H -H ))

8899.0 .0447777 9900.0 .0555555 8888.0 .0339999

LL-A -A rg rgin inin inee

C C H H N N O O 66 11 44 44 22

117744.1 .1111177 117755.1 .1119955 117733.1 .1003399

LL-A -A ssppaara raggin inee

C C H H N N O O 44 88 22 33

113322.0 .0553355 113333.0 .0660088 113311.0 .0446622

LL-A -A ssppaartic rticaaccid id

H H N NO O C C 44 77 44

113333.0 .0337755 113344.0 .0445533 113322.0 .0229977

LL-C -C yysste tein inee

C C H H N NO O SS 33 77 22

112211.0 .0119988 112222.0 .0227700 112200.0 .0112255

C C H H N NO O 55 99 44

114477.0 .0553322 114488.0 .0661100 114466.0 .0445533

LL-G -G lu luta tam m in inee

H H N N O O C C 55 11 00 22 33

114466.0 .0669911 114477.0 .0776644 114455.0 .0661199

G G ly lyccin inee

C C H H N NO O 22 55 22

7755.0 .0332200 7766.0 .0339999 7744.0 .0224422

LL-H -H is istid tidin inee

C C H H N N O O 66 99 33 22

115555.0 .0669955 115566.0 .0777733 115544.0 .0661177

LL-Is -Isoole leuuccin inee

C C H H N N O O 66 11 33 22

113311.0 .0994466 113322.1 .1002255 113300.0 .0886688

LL-L -Leeuuccin inee

C C H H N N O O 66 11 33 22

113311.0 .0994466 113322.1 .1002255 113300.0 .0886688

LL-L -Lyyssin inee

C C H H N N O O 66 11 44 22 22

114466.1 .1005555 114477.1 .1113344 114455.0 .0997777

LL-G -G lu luta tam m ic icaaccid id

LL-M -M eeth thio ionnin inee

C C H H N N O O SS 114499.0 .0551111 115500.0 .0558899 114488.0 .0443322 55 111 22

LL-P -Phheennyyla lala lannin inee

C C H H N N O O 99 111 22

116655.0 .0779900 116666.0 .0886688 116644.0 .0771122

LL-P -Pro rolin linee

C C H H N NO O 55 99 22

111155.0 .0663333 111166.0 .0770066 111144.0 .0665511

LL-S -Seerin rinee

H H N NO O C C 33 77 33

110055.0 .0442266 110066.0 .0550044 110044.0 .0334488

LL-T -Thhre reoonnin inee

C C H H N NO O 44 99 33

111199.0 .0558822 112200.0 .0666611 111188.0 .0550044

LL-T -Tyyro rossin inee

C C H H N N O O 99 111 33

118811.0 .0773399 118822.0 .0881177 118800.0 .0666611

LL-V -Vaalin linee LL-T -Try ryppth thooppaann

H H N N O O C C 55 111 22

111177.0 .0779900 111188.0 .0886688 111166.0 .0771122 220044.0 .0889999 220055.0 .0997777 220033.0 .0882211

C C H H N N 111 12 12 2O 2O 22

Table 1. Amino Acid Standards

Amino Acid

1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6

0.0029

0.003

0.0031

0.0032

0.0033

0.0034

0.0035

0.0036

0.0037

L-Tryptophan L-Leucine L-Phenylalanine L-Isoleucine L-Tyrosine L-Methionine L-Valine L-Aspartic acid L-Glutamic acid L-Alanine

Retention Time 11.07 11.22 11.25 11.31 11.33 11.55 11.61 11.83 11.83 12.05

Amino Acid L-Threonine L-Glycine L-Serine L-Proline L-Asparagine L-Glutamine L-Arginine L-Histidine L-Lysine

Retention Time 12.29 12.32 12.52 12.94 13.20 13.35 16.63 16.67 17.01

0.55 0.5

1/T ( 0 K )

0.45

Figure 6. Vant Hoff Plot With Acetonitrile

1

EEm m ppiric iricaall FFoorm rm uula la C C H H N NO O 33 77 22

p25_02.d x102 +EIC(120.00000-120.20000)ScanMix4_Gradient09A_Tem

Arg

Instrumentation

L-Alanine L-Arginine L-Aspartic acid L-Cystine L-Glutamic acid L-Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tyrosine L-Valine

His

1.200

Pro

Figure 3. Retention Time With Acetonitrile

8

Amino Acid Standards

0.4 0.35 0.3

0

Amino Acid Retention Data Retention time data was collected under isocratic conditions of 50%, 60%, 70%, 75%, 80%, 85% and 90% organic solvent. The organic solvent was composed of 0.1% formic acid in acetonitrile (Figure 3), methanol (Figure 4) or acetone (Figure 5). Water containing 0.1% formic acid made up the difference. The mobile phase flow rate was 0.4 ml/min. The column temperature was 20oC. The column t0 is 1.44 minutes.

40

60

70

80

90

100

% Methanol

Figure 4. Retention Time With Methanol

L-Alanine L-Arginine L-Aspartic acid L-Cystine L-Glutamic acid L-Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tyrosine L-Valine

9 8 7 6 5

Figures 3 -5 are the retention plots for the amino acids investigated in this study. For acetonitrile and acetone there is a substantial increase in retention as the percentage of organic in the mobile phase exceeds 60%. For methanol only a small increase in retention is observed. Thus it appears that aprotic solvents induce greater ANP behavior than protic solvents. Use of acetone which appears to have good ANP properties is possible in LC/MS but not conventional UV detection. Another advantage of the use of ANP is the increase in sensitivity at high organic content in the mobile phase. Typical additives needed to promote ANP retention (acetic or formic acid) are very compatible with mass spectroscopy detection.

50

4 3 2

0 50

60

70

0.25

Retention time reproducibility data was collected for one gradient at 15 and 30oC. The flow rate was 0.4 ml/min and an equilibration time of 5 minutes. The gradient was;

0.15

Time

Retention time vs % acetone in the mobile phase

1

40

Gradient Reproducibility

80

90

100

% Acetone

%B

0

90

5 6

Time

0.2

%B

8

80

90

9

50

80

13

50

Table 2 shows retention time reproducibility for nine amino acids at two temperatures. Four replicates were performed at each temperature. The reproducibility was 0.28% or better for the amino acids. This is a significant improvement over what is usually observed for most HILIC analyses, especially considering this is gradient data with only a 5 minute re-equilibration time between runs.

0.1 0.05 10.5

Lys

Individual amino acid standards were purchased from Sigma-Aldrich. The amino acids studied are in Table 1.

L-Arginine L-Aspartic acid L-Glutamic acid L-Isoleucine L-Asparagine L-Glycine L-Leucine L-Lysine L-Alanine L-Glutamine L-Histidine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tyrosine L-Valine L-Tryptophan

1.400

Gln

90

Asn

Figure 1. Topology Of Silica B And Silica C

% ACN 80

Leu

Figure 2. LC/TOF instrument configuration

O

70

Tyr Met

O

60

Glu Ala Gly Thr Ser

O

50

Asp

O

40

L o g t R (m in )

O

O Si O Si H O O O Si O Si H O O O Si O Si H

0

M SD TOF M SD

Ile

Silica C

M SD TOF

Phe

binary Reg. Gr ad. pum p Pum SLp

O

L-Serine L-Threonine L-Tyrosine L-Valine

3

TCC SL

Figure 7 shows the extracted ion chromatogram (EIC) of a nineteen amino acid mixture under one of the gradient conditions tested. All of the critical amino acid pairs (those that are isobaric or have masses within one mass unit ) are separated under these conditions except for the Leucine / Isoleucine pair. At present, the maximum separation is approximately 0.15 min with 0.30 minutes needed for resolution with the peak widths obtained for these two amino acids. Additional gradient formats and mobile phases are under investigation to address this issue.

Trp

TCC

h-ALS SL

O Si OH O O Si OH O O Si OH

L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline

5

Degasser

Silica B

L-Glutamic acid L-Glycine L-Histidine L-Isoleucine

7

DAD SL

Figure 6 shows the retention behavior of amino acids as a function of temperature (log tR vs. 1/T, vant Hoff plot). In all cases, retention increases as the temperature is increased indicating either a positive enthalpy for interaction of the solute with the stationary phase or substantial entropy contributions (proton activity). This temperature effect is opposite of what is typically observed under reverse phase conditions i.e. decreasing retention with increasing temperature.

Retention time vs. % acetonitrile in the mobile phase

Results and Discussion

Val

A high surface area, 4.0 µm particle size Type-B silica was converted to Type-C material having a silica hydride (Si-H) surface (Figure 1). To improve peak shape of the basic amino acids the Type-C silica was further reacted to reduce residual silanols (